The objectives of the proposed research are to elucidate the mechanism of methemoglobin reduction as it occurs in erythrocytes, to probe the role of proteases in the origin of the components of the methemoglobin reduction system, and to study an erythrocyte green hemeprotein. We will study the chemical and physical properties of the two forms of bovine erythrocyte cytochrome b5. We will attempt to detect, isolate, and characterize the erythroid proteases which convert microsomal forms of cytochrome b5 and cytochrome b5 reductase to the erythrocyte forms of these proteins. The kinetics and mechanism of methemoglobin reduction will be studied in intact cells and in systems reconstituted from purified cytochrome b5, cytochrome b5 reductase, methemoglobin, and NADPH. We will study with a variety of techniques the cytochrome b5 methemoglobin complex that participates in the pathway. We will study methemoglobin reduction in erythrocytes from normal individuals, and from patients with hemoglobin M, congenital deficiency of reductase, and congenital sensitivity to oxidant drugs. We will search for the biological activity of the bovine erythrocyte green hemeprotein. We will determine the structure of the prosthetic group of this protein.